The long-term objective of this proposal is to elucidate the synaptic transmission mechanism. In particular, the role of synaptic vesicles in this process will be investigated. To accomplish this, the temperature-sensitive mutant of Drosophila, shibire-ts1 (shi), which is normal at 18 degree C, but in which endocytosis is blocked at 30 degree C, will be used. In this mutant, recycling of synaptic vesicles is reversibly blocked at 30 degree C, which results in gradual vesicle depletion as exocytosis proceeds uninhibited. This allows precise control of the number of vesicles in a synapse. Taking advantage of this mutant, past obstacles in the physiology of the presynaptic mechanism. e.g., difficulty in the independent determination of number of "available quanta (n)" and their release probability (p) are overcome (in quantum theory, the quantal release of transmitter (n) is assumed to be governed by the statistical release of "available quanta (n)" with the probability of p; i.e., m = pn). These quantal parameters (n and p) which are the theoretical basis of the quantum theory, will be determined by a combination of physiological experiments and electronmicroscopic observations. Then, a morphological correlate (e.g., vesicles or active sites) for n will be determined using electron microscopy. Our preliminary data suggests that n may represent a subpopulation of releasable vesicles. The time course of mobilization of vesicles into the releasable condition will be calculated. Also, the relationship between subminiature excitatory junction potentials (sub-m.e.j.p.'s), classical m.e.j.p.'s, and vesicles will be determined by varying the number of vesicles in the terminal and correlating this with frequency and amplitude changes in the sub-m.e.j.p. and m.e.j.p. populations. A morphological correlate for sub-m.e.j.p.'s will also be sought. The mechanism responsible for the "clustering" of m.e.j.p. release, which results in multiquantal or "giant" m.e.j.p.'s during recovery from depletion, will be investigated by varying the external Ca++/Mg++ ratio. A morphological correlate for this phenomenon will be sought. Also, a morphological correlate for a releasable subpopulation of synaptic vesicles associated with the presynaptic dense body will be investigated using electron microscopy, and the process of exocytosis itself will be morphologically characterized. The long-term effect of vesicle depletion on the synapse and innervated muscle will also be investigated. By furthering our understanding of synaptic transmission, it is hoped that this research will provide a basis for the diagnosis and treatment of neurological disorders.